(375g) Insights for the Engineering of More Efficient Inteins | AIChE

(375g) Insights for the Engineering of More Efficient Inteins

Authors 

Pereira, B. - Presenter, Rensselaer Polytechnic Institute
Shemella, P. - Presenter, Rensselaer Polytechnic Institute
Garde, S. - Presenter, Rensselaer Polytechnic Institute
Belfort, G. - Presenter, Rensselaer Polytechnic Institute
Nayak, S. - Presenter, Rensselaer Polytechnic Institute
Belfort, M. - Presenter, Wadsworth Center, NYS Department of Health


Inteins are intervening protein segments that mediate the splicing of flanking protein sequences, termed exteins. This protein splicing mechanism consists of four reactions which together results in the cleavage of two peptide bonds at the intein's N and C termini and the formation of a peptide bond between the exteins. However, cleavage at only one terminus may occur, yielding side products and inefficient splicing. In some applications, the N- or C-terminal cleavage is desired and achieved by altering the splicing reaction mechanism through mutagenesis. Through experimental methods such as site-directed mutagenesis of conserved residues and subsequent assays for intein activity, as well as computational methods including first principles density functional theory and combined quantum mechanics and classical molecular mechanics (QM/MM), we have investigated the reactions of protein splicing. These results indicate that certain mutations either inhibit or enhance specific reaction steps of the overall splicing mechanism, providing insight into how inteins can be engineered for more efficient activity.